Ultrasound Stimulates Ethanol Production during the SimultaneousSaccharification and Fermentation of Mixed Waste Office Paper

B. E. Wood, H. C. Aldrich, and L. O. Ingram*

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida,Gainesville, Florida 32611

The commercial production of ethanol from cellulose by simultaneous saccharificationand fermentation (SSF) is prevented in part by the high cost of fungal cellulaseenzymes. Intermittent exposure of SSF processes to ultrasonic energy under selectedconditions (5 FPU of cellulase/g of substrate; 15 min of exposure/240 min cycle duringthe latter half of SSF) was found to increase ethanol production from mixed wasteoffice paper by approximately 20%, producing 36.6 g/L ethanol after 96 h (70% of themaximum theoretical yield). Without ultrasound, 10 FPU of cellulase/g of substratewas required to achieve similar results. Continuous exposure of the organism toultrasonic energy was bacteriostatic and decreased ethanol production but may beuseful for the controlling bacterial growth in other processes.

Introduction

The bioconversion of soluble sugars derived from wastepaper represents a potential opportunity for the renew-able production of organic chemicals such as ethanol(Kerstetter and Lyons, 1991). The high cost associatedwith cellulose hydrolysis, however, remains a majorproblem (Wyman, 1994). Although concentrated acid hasbeen used in past wood-to-ethanol processes (Harris etal., 1946; Katzen and Othmer, 1942; Leonard and Hajny,1945; Jacobs, 1950), theoretical yields are limited by thisapproach and implementation may require the develop-ment of new, cost-effective methods for acid recovery andreuse (Wyman, 1994). Enzymatic hydrolysis provides anenvironmentally friendly means of depolymerizing cel-lulose and the potential for higher yields, but costs arealso unfavorable. In addition, cellulases are subject tostrong end-product inhibition by soluble products suchas cellobiose and glucose (Grohman and Himmel, 1991).Gauss et al. (1976) developed a yeast-based simultaneoussaccharification and fermentation (SSF) process whichsubstantially reduced inhibition by glucose. With indus-trial yeasts (Saccharomyces), however, large amounts ofsupplemental â-glucosidase are required to prevent theaccumulation of cellobiose. Modifications of this originalSSF process remain the best available technology forcellulose conversion to ethanol.The requirement for supplemental â-glucosidase was

eliminated by the genetic engineering of Klebsiella oxy-toca strain P2, a bacterium which contains a high-affinitycellobiose uptake system (Lai et al., 1997; Wood andIngram, 1991). With this organism, cellulose conversionby SSF was optimal at pH 5.0-5.2 and 35-37 °C (Doranand Ingram, 1993), conditions which are favorable for theactivities and stability of fungal cellulases. The continu-ous removal of cellobiose and glucose during SSF withK. oxytoca P2 eliminates the inhibition of cellulase bysoluble products.Previous studies have shown that the rate of cellulose

hydrolysis declines during SSF processes despite the

abundance of excess substrate and the stability of theenzymes (Kohlmann et al., 1995; Nidetzky and Steiner,1993; Philippidis and Smith, 1995). This decline appearsto result from nonproductive associations of cellulase withsubstrate, which can be re-activated by a mild heattreatment (Brooks and Ingram, 1995; Doran et al., 1994).Gusakov and colleagues (1996) have shown that intensiveagitation also increases the efficiency of cellulose hy-drolysis. Like heat treatments, intensive agitation mayfacilitate enzyme dissociation and rebinding at siteswhich are productive for hydrolysis.Recent studies demonstrating that ultrasound can be

used as an environmentally benign method to enhancethe deinking of recycled paper also reported that fiberstructure was altered by this treatment (Scott andGerber, 1995; Sell et al., 1995; Norman et al., 1994).Ultrasonic treatment increased the water-holding capac-ity of pulp (decrease in “freeness”) by increasing theabundance of capillary-like regions which retard drain-age. Such changes in fiber structure may be beneficialfor the bioconversion of cellulose to ethanol.In this study, we have investigated the effects of

ultrasonic treatments on fiber structure at the ultra-structural level and on the SSF process with fungalenzymes using K. oxytoca P2 as the biocatalyst.

Materials and Methods

Organism andMedia. K. oxytoca P2 was used as thebiocatalyst in all fermentations. Seed cultures weregrown for 16 h at 30 °C without agitation in Luria broth(LB) (5 g/L yeast extract, 10 g/L tryptone, and 5 g/L NaCl)containing 50 g/L glucose (Brooks and Ingram, 1995).Stock cultures were maintained on solid LB medium (15g/L agar, 20 g/L glucose, and 0.6 g/L chloramphenicol).Materials. Spezyme CP (100 filter paper units (FPU)/

mL; Genencor International, South San Francisco, CA)was used as a source of fungal cellulase (Trichodermalongibrachiatum). Novozyme 188 (250 IU/mL; Novo-Nordisk, Franklinton, NC) was used as a source ofsupplemental â-glucosidase (Aspergillus niger) duringsaccharification experiments. Both enzyme preparationswere generously provided by the manufacturers. Car-boxymethylcellulose and p-nitrophenyl-â-D-glucopyrano-side (p-NPG) were purchased from the Sigma Chemical

* Send correspondence to Lonnie O. Ingram, Department Mi-crobiology and Cell Science, IFAS, P.O. Box 110700, Universityof Florida, Gainesville, FL 32611. Phone: (352) 392-8176. FAX:(352) 846-0969. E-mail: lingram@micro.ifas.ufl.edu.

232 Biotechnol. Prog. 1997, 13, 232−237

S8756-7938(97)00027-1 CCC: $14.00 © 1997 American Chemical Society and American Institute of Chemical Engineers

Co. (St. Louis, MO). Yeast extract and tryptone wereproducts of Difco (Detroit, MI).Mixed waste office paper (MWOP) was used as the

substrate for bioconversion. Two batches were obtainedfrom two local offices. MWOP contains approximately90% carbohydrate and 10% inert materials (maximumtheoretical yield, 0.90 × 0.568 ) 0.51 g of ethanol/g ofMWOP).Modification of New Brunswick Multiferm Fer-

mentor. New Brunswick Model 100 Multiferm fermen-tors (Edison, NJ) (14-L vessel, 10-L working volume) weremodified for use with MWOP (Figure 1). The thermistorwell, heat exchangers, and baffles were removed from thehead plate to facilitate improved mixing. The condenserwas removed to allow insertion of an ultrasonic probethrough the head plate. The thermometer well was alsopartially removed. Mixing was provided by two Rushton-type radial flow impellers (75-mm diameter), a marineimpeller (68-mm diameter), and a wire scraper (attacheddirectly to the lower radial impeller using small stainlesssteel bolts). The combination of impellers providedmixing and directional flow; the wire scraper minimizedsettling of pulp (a problem only in the earliest stages ofSSF).Temperature was controlled using an external water

jacket, supported by an adjustable laboratory jack.Height adjustment was essential to allow insertion andremoval of the fermentation vessel. The water jacket wasconstructed by lining the inside of a 20-L Nalgene #54102-0005 polyethylene cylinder (Rochester, NY) witha 2 m copper coil (1/4 in. od). Water was added above thelevel of the fermentation broth. The controlling andmeasuring thermistors were inserted into the waterjacket and head plate, respectively. The copper coil wasconnected to the Microferm temperature-control system(recirculating). The external bath was as effective as theoriginal New Brunswick design.Ultrasonic Treatment. Ultrasound was generated

using a Telsonic 36 kHz tube resonator (>95% efficiency),model RS-36-30-1, with an accompanying model MRG-36-150 (constant 150-W effective output) ultrasonicgenerator (Bridgeport, NJ). Frequency was tuned auto-matically. Treatment times were controlled using nestedSPER Scientific 810030 electronic timers (Fisher Scien-tific Co., Norwalk, GA).Assembly and Operation of the 14-L Fermentor.

Head plates were sanitized by coating all surfaces with

aqueous formaldehyde (10 g/L) while loosely enclosed ina large plastic bag. Formaldehyde was allowed to dis-sipate for 24 h before assembly. After processing withan office paper shredder, 1 kg of MWOP (dry weight) wasplaced in a 14-L fermentation vessel; 8 L of H2O contain-ing 110 mL of 18 N H2SO4 (to neutralize carbonates) wasadded and the suspension autoclaved for 1 h (120 °C).The paper mixture was then homogenized using a handdrill fitted with a paint-mixing blade, autoclaved for 1h, and allowed to cool overnight (pH 4.5-5.0). Using alarge baking whisk (sterile), the pulp was mixed with 50mL of Spezyme CP (5 FPU/g MWOP), 1 L of sterile 10X-concentrated LB (adjusted to pH 5.0), and sufficientsterile distilled water to make 10 L.Seed cultures were harvested by centrifugation (5000g)

and added as an inoculum (165 mg/L dry weight).Fermentations were conducted at 35 °C without pHadjustment (approximately pH 5.0; 200 rpm). Durationof exposure to ultrasound was varied during SSF.Samples were removed for the determination of pH andethanol at 24-h intervals.Cell Viability. For viability studies, a 2-L stainless

steel beaker containing 1.75 L of LB (50 g/L glucose and40 mg/L chloramphenicol) was inoculated with K. oxytocaP2 to an initial density of 165 mg/L (0.5 OD550nm). Thebeaker was immersed in a water bath at 35 °C andcontinuously stirred (120 rpm). Incubation was contin-ued for 12 h with or without ultrasonic treatment.Samples were removed and diluted appropriately todetermine viable cell number as colony-forming units(CFU) and cell mass (OD550nm). Broth pH was alsomonitored.Saccharification of MWOP. Pulp was prepared and

autoclaved as described for fermentation. Cooled pulpwas diluted to a final concentration of 50 g/L (dry weight)by adding citrate buffer (50 mM final concentration), 2.5mL of Spezyme CP/L (5 FPU/g of MWOP), 0.4 mL ofNovozyme 188/L (2 IU/g of MWOP), and DW. Whenneeded, a small amount of HCl or NaOH was added toachieve pH 5.2. Thymol (0.5 g/L) and chloramphenicol(40 mg/L) were added to prevent microbial growth. Theresulting slurry (2.5 L) was placed in a 3-L stainless steelbeaker and incubated at 35 °C with continuous agitationusing a Ciamfanco model BDC-1850 laboratory mixer(Fisher Scientific Co.) equipped with a Rushton impeller(75-mm diameter). Stirrer speed was periodically re-

Figure 1. Diagram illustrating modification of a New Brunswick Scientific Multiferm 100 and head plate for use with paper pulp.

Biotechnol. Prog., 1997, Vol. 13, No. 3 233

adjusted to the lowest setting that allowed mixing.Samples were removed for the determination of reducingsugars.Enzyme Stability. Enzyme preparations were di-

luted in 50 mM citrate buffer to concentrations equivalentto those used in the study of sugar release from MWOP,250 FPU/L cellulase (Spezyme CP) and 100 IU/L â-glu-cosidase (Novozyme 188). Thymol and chloramphenicolwere added to prevent microbial growth. The enzymepreparation was mixed continuously (120 rpm) duringincubation at 35 °C, with or without ultrasonic treatment.Samples were removed to measure endoglucanase andâ-glucosidase activities.Enzyme Assays and Analyses. Appropriately di-

luted samples were assayed for endoglucanase activityat 35 °C (pH 5.2) using 20 g/L low-viscosity carboxy-methylcellulose as a substrate (Wood and Bhat, 1988).Reducing sugars were measured using the dinitrosalicylicacid method (Chaplin, 1986). Cellobiase (â-glucosidase)activity was determined by measuring p-NPG hydrolysis(Wood and Bhat, 1988). Appropriately diluted enzymesamples were assayed at 35 °C in 50 mM citrate buffer(pH 5.2) containing 2 mM p-NPG. Reactions wereterminated by adding an equal volume of 1 M Na2CO3.The absorbance of p-nitrophenol was measured at 410nm. Ethanol was determined by gas-liquid chromatog-raphy using n-propyl alcohol as an internal standard(Brooks and Ingram, 1995).Ultrastructural Observations. The fiber structure

in MWOPwas examined using a Hitachi S-4000 scanningelectron microscope (Danbury, CT). Dried samples weresputter-coated with gold to prevent charging (Doran etal., 1994).

ResultsEffect of Ultrasonic Treatment on Ethanol Pro-

duction by SSF. Control experiments were conductedwithout ultrasonic treatment using two concentrationsof cellulase (Table 1). Ethanol production with 10 FPU/gMWOP (1000 FPU/L) was 20%-60% higher than with 5FPU/g MWOP (500 FPU/L), indicating that saccharifi-cation is limiting at the lower enzyme level. The effectof ultrasonic treatment was investigated using only thelower enzyme concentration (5 FPU/g MWOP).During SSF, treatment with ultrasound for 15 min/

240-min cycle was almost as effective as doubling thelevel of cellulase (Table 1, Paper Batch I). Ultrasonictreatment caused a 50% increase in ethanol productionafter 24 h and a 16% increase in ethanol yield after 96h. The 15 min/240-min cycle regimen appears to ap-proach the optimum. Lower ethanol concentrations were

obtained with shorter but more frequent treatments andwith longer treatments.A second set of experiments (Paper Batch II) was

conducted to evaluate the benefit of ultrasonic treatment(15 min/240-min cycle) during each half of the SSFprocess (Table 1). The highest levels of ethanol wereproduced when treatment with ultrasound was limitedto the latter half of the SSF process. This treatmentincreased ethanol production by 20% to 36.6 g/L after 96h, equivalent to 70% of the maximum theoretical yieldfor MWOP. Treatment during the first 48 h resulted inan initial increase in ethanol production, but productiondeclined when treatments were terminated.Uninterrupted exposure to ultrasound was detrimental

to the SSF process (Table 1). With continuous ultra-sound, no stimulation in ethanol production was observedand fermentation appeared to end after 24 h. Thesensitivity of the SSF process to the scheduling ofultrasonic treatments was surprising. To understand thebasis for this phenomenon, further studies were con-ducted which evaluated the effects of ultrasound onindividual components of the SSF process: enzymestability, saccharification, cellulose structure, growth,and viability of the biocatalyst.Enzyme Stability. The decline in ethanol production

during continuous exposure to ultrasound does not resultfrom inactivation of fungal enzymes (Figure 2). Fungalendoglucanase and cellobiase activities remained stableover the 48-h period examined. Surprisingly, cellobiaseactivity increased by approximately 40% during the first24 h of incubation at 35 °C then declined to near theinitial value after 48 h. Endoglucanase activity remainedconstant for 24 h and then declined slightly. The basisfor the transient increase in cellobiase activity is un-

Table 1. Effects of Ultrasonic Treatment on Ethanol Production from MWOP

ethanol (g/L)ultrasonic treatment replicates

enzymea(FPU/g of MWOP) 24 h 48 h 72 h 96 h

Paper Batch Inone 2 10 15.7 27.3 33.5 35.3none 4 5 9.5 ( 2.3 19.0 ( 2.7 25.7 ( 2.5 29.4 ( 2.9b15 min/240-min cycle 5 5 14.3 ( 2.0 26.1 ( 1.3 31.3 ( 1.3 34.0 ( 1.9b15 min/120-min cycle 2 5 13.4 23.4 28.8 31.47 min/240-min cycle 1 5 13.2 20.3 26.8 29.660 min/240-min cycle 1 5 14.5 21.7 22.9 22.2continuous 2 5 10.2 11.2 11.3 11.3

Paper Batch IINone 2 5 11.4 21.8 28.2 30.715 min/240-min cyclec (0-48 h) 2 5 13.0 24.2 27.8 28.615 min/240-min cyclec (48-96 h) 2 5 16.3 25.3 33.1 36.6a FPU of spezyme CP cellulase added per gram of MWOP. SSF broth contained 100 g of MWOP/L. b All ethanol values for the ultrasonic

treatment, 15 min/240-min cycle, were judged to be significantly higher than the untreated control at 24, 48, 72, and 96 h (unpairedt-test, p ) 0.001-0.018). c Ultrasonic treatment during the first half (0-48 h) or last half (48-120 h) of the SSF experiment.

Figure 2. Effect of ultrasound on the stability of commercialendoglucanase and cellobiase activities. Results are expressedas a percentage of total activity measured prior to ultrasonictreatement.

234 Biotechnol. Prog., 1997, Vol. 13, No. 3

known but may be due to dispersal of protein aggregatesoriginally present in the concentrated, commercial prepa-rations.Saccharification. Ultrasonic treatment increased

the rate of saccharification of MWOP above that of thecontrol in all cases (Table 2). This increase in reducingsugars was due to an enzyme-mediated process ratherthan physical or chemical action by reactive productsfrom the sonolysis. Without added enzymes, the low levelof reducing sugar initially present did not increase during48 h of incubation with continuous exposure to ultra-sound.

Periodic exposure to ultrasound was more beneficialfor saccharification than continuous exposure, analogousto the complete SSF process. Exposure to ultrasound for15 min/120-min cycle increased the rate of saccharifica-tion by 30-40% throughout the 48-h period. Increasedhydrolysis was roughly proportional to total ultrasonicenergy input, with an optimum of around 1.8 kWh/96 hof SSF.Ultrastructure of MWOP. The energy dependence

of intermittent ultrasonic treatment on MWOP saccha-rification suggested that physical changes in the sub-strate may be involved. The fine structure of samplesfrom the saccharification experiments was examinedafter incubation for 4 h with and without enzymes andafter 1 h of continuous ultrasonic treatment. At lowmagnification, all appeared essentially identical, indicat-ing that overall fiber dimensions had not been altered(Figure 3A). However, significant differences were ob-served at high magnifications (Figure 3 B-D). Enzymetreatment caused a reduction in angular regions andappeared to polish the surface (Doran et al., 1994).Ultrasonic treatment converted the fiber surface into atangle of filaments, each 20-40 nm in diameter andmany microns in length. These filaments are similar insize to microfibrils produced by the cellulose biosyntheticcomplex in plant cell membranes (Brett and Waldron,1996), the native constituents of cellulose fibers. Al-though the disruption of fiber structure by ultrasound

Table 2. Effects of Ultrasound on EnzymaticSaccharification of MWOP

glucose equivalentsc (mM)ultrasonictreatmenta

no. ofbreplicates 24 h 36 h 48 h

none 3 88.1 ( 6.1 98.3 ( 6.1 106.9 ( 7.815 min/240-min 3 96.5 ( 6.4 113.6 ( 8.0* 128.3 ( 8.4*15 min/120-min 3 115.5 ( 14.3* 133.2 ( 11* 149.0 ( 11*continuous 3 98.1 ( 2.5* 112.5 ( 5.5* 126.6 ( 2.1*continuous

(no enzyme)2 0.67 0.67 0.58

a Ultrasonic treatments were automatically controlled at thestated intervals. b All experiments contained 5 FPU of SpezymeCP and 10 IU of Novozyme 188/g MWOP, except the control whichlacked both enzymes. c Sugar values for ultrasonic treatmentswhich were judged to be significantly higher than the controlwithout ultrasound (unpaired t-test, p ) 0.006-0.058) are markedby an asterisk.

Figure 3. Ultrastructure of treated and control MWOP: (A and B) MWOP after 1 h of ultrasonic treatment; (C) untreated MWOP;(D) MWOP after 4 h of cellulase treatment.

Biotechnol. Prog., 1997, Vol. 13, No. 3 235

appears to be limited to the surface, it is possible thatother microfibrils have been stripped from the surfaceduring treatment. Dissociation of component microfibrilsby ultrasound would be expected to expose new areas forcellulase attack, consistent with the observed increasesin the rate of saccharification.Growth and Viability of the Biocatalyst. The

susceptibility of K. oxytoca P2 to ultrasonic damage wasinvestigated as a possible basis for the detrimental effectof continuous exposure to ultrasound. As shown inFigure 4, a classical growth curve was observed for P2during incubation in LB (plus glucose) without ultra-sound. Continuous exposure to the ultrasonic energylevel used in SSF experiments was nonlethal but ef-fectively inhibited cell division (CFU) and increases incell mass (OD550nm). Growing cells acidify the broth byproducing CO2 and small amounts of organic acids. Thesmall rise in broth pH observed for cells continuouslyexposed to ultrasound indicates that ultrasonic treatmentcaused sufficient damage to prevent the fermentation ofsugars.

DiscussionThe bioconversion of cellulose to ethanol by SSF is

limited by the cost of cellulase enzymes required forsaccharification. The beneficial action of ultrasound ona cellulase-limited SSF process appears to result from acombination of effects on substrate structure and enzy-matic saccharification. Ultrasonic cavitation is a complexand dynamic phenomenon which is influenced by manyfactors including temperature, dissolved gasses, sus-pended particulates, proximity of the resonator to thenucleating surface, etc. (Atchley and Crum, 1988; Price,1992; Shoh, 1988). Accessibility of the substrate tocellulase is a primary factor which limits the efficiencyof enzymatic hydrolysis (Kohlmann et al., 1995; Philip-pidis and Smith, 1995; Nazhad et al., 1995). In our study,the intense but minute areas of energy foci produced byultrasonic cavitation disassembled the relatively smoothsurface of cellulose fibers into component microfibrils,increasing the hydrated surface area available for enzy-

matic attack. Fungal cellulase enzymes bind tightly tocellulose and appear to become progressively trapped atsites which are nonproductive for hydrolysis (Brooks andIngram, 1995; Doran et al., 1994; Tomme et al., 1995).The extent of cellulase binding has been shown to bedependent on the intensity of agitation (Kaya et al.,1994). Thus it is likely that the high intensity mixingat the particle surface caused by ultrasound also in-creases the dissociation of cellulase and thus allows activeenzymes to rebind at new sites which are productive forcontinued hydrolysis. Analogous effects on both surfacestructure and enzyme dissociation may also occur in theintensive mass transfer reactor described recently whichachieved extremely high rates of cellulose hydrolysis(Gusakov et al., 1996).Surprisingly, discontinuous ultrasonic treatments were

more beneficial for saccharification than continuousexposure. Continuous exposure did not destroy cellulaseactivity, and this treatment would be expected to pro-gressively increase the hydrated surface area availablefor hydrolysis. The basis for this detrimental effectappears to reside in both the hydrolysis process and thesensitivity of the biocatalysts. Continuous ultrasoundmay decrease cellulase binding to such an extent thatcatalysis is impaired.Continuous exposure to ultrasound was not lethal for

K. oxytoca P2 but effectively inhibited sugar metabolism,growth, and cell division. This inhibition may result fromincreased leakage of intracellular metabolites, inductionof SOS response proteins as shown by Volmer et al.(1996), and other more direct effects on catalysis. Theoptimal regimen for saccharification alone, 15 min ofultrasound/120-min period, was surprisingly similar tothe optimal regimen for the complete biological SSFprocess, 15 min of ultrasound/240-min period. The moreextended period without ultrasound may be required forthe biocatalyst to repair cell damage and resume fer-mentation.The use of ultrasound in the conversion of cellulose to

ethanol may represent a significant improvement in theSSF process. However, projected costs for electricalenergy are high without further optimization of treat-ment cycles and volumes. Our best ultrasound regimenduring SSF utilized 0.45 kWh of electricity and produced366 g of ethanol (10 L). Thus approximately 1.0 kWh ofenergy was required to produce each liter of ethanol (800g). At a cost of $0.06/kWh, energy cost for ultrasonictreatment alone would be $0.06/L of ethanol which mustbe added to other process costs. However, ultrasound canalso be generated by alternative methods such as a liquidwhistle (Price, 1992; Shoh, 1988; Scott and Gerber, 1995).While unable to produce energies which catalyze chemi-cal changes, liquid whistle systems have been used bythe paper industry to increase the water holding capacityof recycled pulp (Scott and Gerber, 1995). With such adevice fitted into a recycle loop used for fermentoragitation, it may be possible to achieve the beneficialaction of ultrasound (disruption of cellulose fine structureand increased saccharification) with a minimum of ad-ditional cost.

Acknowledgment

The authors gratefully acknowledge Scott Whittaker,Donna Williams, and the Interdisciplinary Center forBiotechnology Research for assistance with electronmicroscopy. This research was supported by the FloridaAgricultural Experiment Station (Publication NumberR-05475) and grants from the U.S. Department ofAgriculture, National Research Initiative (95-37308-

Figure 4. Effects of ultrasound on the growth and survival ofK. oxytoca P2: (A) cell mass as OD550nm and broth pH; (B) cellviability as colony-forming units (CFU).

236 Biotechnol. Prog., 1997, Vol. 13, No. 3

1843), and the U.S. Department of Energy, Office of BasicEnergy Science (DE-FG02-96ER20222).

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Accepted March 17, 1997.X

BP970027V

X Abstract published in Advance ACS Abstracts, May 1, 1997.

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